Accurate measurement of glutathione for disease diagnosis and drug metabolite screening

ABSTRACT

A method of measuring and calculating (preferably by a computer with output to a user) tGSH (total glutathione very particularly defined) with sample preparation and assay methods that have been confirmed to provide accurate and reliable tGSH and related diagnostic assays in blood or tissue from a patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to, and incorporates herein byreference, U.S. Provisional Patent Application No. 61/550,656 filed 24Jun. 2011.

BACKGROUND OF THE INVENTION Field of the Invention

Measurement of total Glutathione in blood and other body fluids andtissues must be accurate in order to have diagnostic significance or theability to participate in drug metabolite screening.

Description of Related Art

Previously, glutathione deficiency was examined as a possible prognosticfor survival in Acquired Immunodeficiency Syndrome (AIDS), as disclosedfor example in U.S. Pat. No. 5,843,785. In that patent, generalglutathione levels of whole blood and/or T-cells of HIV positivepatients were determined (directly or indirectly) as an indication ofthe likely period of survival, as well as the need for agents forenhancing the glutathione levels. But the determination made was notaccurate enough to make any kind of correlation with survival expectancyof HIV positive patients. Moreover, it was also mentioned “referring toglutathione levels, it will be understood that it is the valuedetermined by an assay which can be used for comparisons, but does notprovide an absolute value”. Therefore since the glutathionedeterminations were not accurate and absolute, the staled hypothesis wasnot valid enough to corroborate probability of survival in HIV positivepatients. The probability of survival expectancy of HIV positivepatients does not depend on one particular biomolecule measurement suchas glutathione, but on several other conditions associated with theetiology of the disease. The methods both (HPLC and FACS) used for thedetermination glutathione levels are not robust enough to address theproblems associated with quantification like reduced glutathioneoxidation, and conjugates formation.

Similarly, in Pastore, G. Federici, E. Bertini, F. Piemonte, “Analysisof glutathione: implication in redox detoxification,” Clinical Chim Acta333 (2003) 19-39, blood concentrations of reduced glutathione (GSH) invarious pathologic conditions, and the statistical significance of thepurported validation one or more sub-species, are indicated as beingdiagnostically significant. Unfortunately, most if not all of theasserted data are not accurate, which presents a significant problem forphysicians in assessing and treating patients. In many cases theimprecision of these GSH and GSSG numbers overlap as they are notcompared with confidence intervals but with means which are notstatistically different.

United States Published Patent Application No. US2011/0144205 A1hypothesized that glutathione is a key biomarker for heart failureasymptomatic patients, stating that “inflammation and oxidative stressare key components of in the pathophysiology and progression of heartfailure and are strongly associated with the disease severity.”Chemically, oxidative stress is associated with increased production ofoxidizing species or a significant decrease in the effectiveness ofantioxidant defenses. Therefore, these antioxidant defenses can be notonly glutathione but also can be vitamin E, vitamin C, enzymes likesuperoxide dismutase, metallothionein, etc. The method forquantification of glutathione mentioned is not accurate and preciseenough to correct for glutathione oxidation and conjugate formations.

In “Methods for the Quantitation of Oxidized Glutathione,” U.S. Pat. No.6,235,495 B1, the method used for quantification of oxidized glutathionebased on the assay developed by Tietze, which is based onspectrophotometric determination of the reaction of the Ellman's reagentwith Glutathione disulfide. The observation is made by assessing thecolor at a particular absorbance. The invention mentioned in this patentis the use of 1-methyl-2-vinylpyridinium trifluromethanesulfonate (M2VP)to prevent the conversion of GSH to GSSG without interfering withglutathione reductase in the biological sample. However, this method isnot accurate and precise enough to make since clinical and therapeuticdecisions, it is based on the color change at particular absorbance andany other disulfide present in the biological sample can interfere withthe process and can lead to false positive result.

The invention in the published U.S. Patent Application No.US2009/0029409 involves determination of oxidative stress biomarker,which can be varied by perturbation of glutathione levels in the blood.The oxidative stress biomarker candidate identified is ophthalmic acid,but the mechanism as to how glutathione is affected has not beenestablished clearly. Although reduced glutathione is present in lowconcentrations and prone to oxidation in blood, it still can bequantified by using NEM in the sample preparation which minimizes theconversion of reduced glutathione to oxidized glutathione. The normalrange of ophthalmic acid present in the blood has not been mentioned andwhether its production is varied by any other biomolecule otherglutathione has not been discussed.

Zhenying Yan and coinventors have described, in two US patents,2005/US0287623 and 2011/US788186, a method for detecting reactivemetabolites by isotope trapping with a mass spectrometer for the purposeof toxicological assessment. In both Yan patents, the European patent byMichael J. Avery (EP, 1,150,120, October 2001) has been provided as aprior art that involves incubating a test compound with a microsomaldrug metabolizing enzyme system in the presence of a form of glutathioneand then, detecting certain glutathione adducts formed therefrom usingtandem mass spectrometry. Yan points out that Avery method will identifyreactive metabolites as well as non-reactive components (including bothunreactive and metabolites and components of the reaction mixture)formed as a result of common response in mass spectrometry detection,thus resulting in false positives. In the 2005-Yan patent, a method ofdetecting reactive metabolites of a drug candidate whereby the drugcandidate was mixed with a non-labeled trapping agent, an isotopicallylabeled trapping agent and an enzyme. Purportedly improving on the2005-patent, Yan and Norman D Huebert, U.S. Pat. No. 7,884,186 B2,disclosed in the 2011 text that the 2005-patent detects only “soft”metabolites, but does not simultaneously detect both “hard” ad “soft”reactive metabolites. In the 2011 Yan specification, the “soft”metabolite is described as any electrophilic metabolite which compriseat least one substituent group which readily reacts with softelectrophiles, such as the sulfhydryl group in cysteine and the —SHgroup on the compound of the formula. Disclosed examples of softmetabolites were given as quinones, quinone imines, immunoquinone,methids, epoxides, arene oxides and nitrenium ions. The same patentdescribed a hard metabolite as an electrophilic metabolite whichcomprises at least one substitute group which readily reacts with the—(CH₂)₄—NH₂ of the compound of the formula. The disclosed example ofsuch substituent group was given as aldehydes. Additionally, the samepatent claims that its isotope trapping technique was capable ofeliminating false positives using mass spectrometric patternrecognition. There is no indisputable evidence in either of the Yanpatents that would support the claims associated with the “eliminationof false positives.” Without standard reference materials, amathematical basis and statistical support, such claims would not meetthe criteria established for mission critical applications such as thoseestablished in the clinical, diagnostic and homeland security fields.

In the above exemplary prior art, the field attempts to identify variousglutathione adducts and conjugates has therefore been fraught withfundamental problems. A need remains, therefore, for an accurate methodof measuring and calculating tGSH and specific related compounds andconjugates in a patient's blood, fluid or tissue sample and theconcomitant diagnostic method of interpreting the measurements andratios for diagnostic and prognostic purposes. A need also remains forimproved accuracy in situ toxicological drug screening of biologic drugsinvolving GSH, GSH species, GSH conjugates, and GSH metabolites.

SUMMARY OF THE INVENTION

In order to meet this need, the present invention is a method ofmeasuring and calculating (by a computer with output to a user) tGSH(total glutathione very particularly defined) with sample preparationand assay methods that have been confirmed to provide reliable tGSH andrelated diagnostic assays in blood or tissue from a patient. Moreover,the method of accurate measurement and calculation of tGSH, GSH (reducedglutathione) and the ratio of GSH to oxidized glutathione (GSSG) hasenabled for the first time the identification of the diagnostic range ofthe ratio of GSH/GSSG as 15-200, where in a human having a ratio withinthe range 15-200 is vulnerable to disease including but not limited toautism and chronic fatigue syndrome. An increase in this ratio overtreatment time for a patient indicates positive response to therapy—upto maximum ratio values of about 600-800 for healthy individuals. Inparticular, after accurate measurement and calculation according to thisspecification, a tGSH concentration of 2.8-3.0 micromoles tGSH per gramof blood is prognostic of autism in children and young adults, and a GSHlevel of 2.2-2.4 micromoles per gram of blood is prognostic of autism inchildren and young adults. The measurement are conducted using speciatedisotope dilution mass spectrometry to assess particular variants(reduce, oxidized, conjugates, etc.) of glutathione and the calculationsare preferably conducted automatically, following mass spectrometry, toprovide readouts of tGSH, GSH and the ratio of GSH/GSSG using thefollowing equations, where tGSH is the sum of GSH_(corrected),GSSG_(corrected) and CGSH, and the ratio is given by:

$\frac{GSH}{GSSG} = \frac{z}{\gamma}$

where:z=GSH_(corrected)=GSH_(SIDMS)+α+βα=[GSH

GSSG]β=CGSH=Σ_(i)CGSH₁+CGSH₂+ . . . CGSH_(i)γ=GSSG_(corrected) (GSSG−[GSH

GSSG])The mass spectrometry measurement and calculations are preferablyperformed using a computer and the results are presented as an output toa user, usually a printout on paper or a readout on a computer screen,showing values for tGSH, GSH and the ratio of GSH/GSSG.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIG. 1: A bar graph showing prior art literature values for ratio oftotal glutathione to reduced glutathione;

FIG. 2: A line graph showing average red blood cell tGSH according tothe invention, in children with autism and controls. The p value is0.011 with n=27 pairs.

FIG. 3: A line graph showing mean red blood cell GSH concentrations inchildren with autism and controls. For n=25 pairs, the results aresignificant with a p value of 0.0032.

FIG. 4: A line graph showing red blood cell tGSH concentration times redblood cell GSH in children with autism and controls. For n=25 pairs, thep value equals 0.0042.

FIG. 5: A line graph showing logistic regression analysis of red bloodcell tGSH concentration versus autism diagnosis probability for n=27pairs.

FIG. 6: A line graph showing Logistic regression analysis of red bloodcell GSH concentration versus autism diagnosis probability for n=25pairs.

FIGS. 7a and 7b : Mass spectrometry printouts of simultaneous analysisof glutathione conjugated with three mercury species by D-SIDMS usingnano-ESI-TOF-MS.

FIG. 8: A bar graph shows the GSH/GSSG corrected ratios of two youngtwin female ASD patients. The n approximately 12 replicate measurementswith 95% confidence limit.

DETAILED DESCRIPTION OF THE INVENTION

Quantitatively low, high, and quantifiable levels of reduced andoxidized glutathione species (GSH and GSSG, respectively) and conjugatesof glutathione are diagnostic and prognostic of diseases including butnot limited to immune system diseases and autism. The invention is amethod of accurately and reliably assaying for particularly definedglutathione species levels, together with quantitative interpretation ofthe data for diagnostic significance. The method applies direct,mathematical, calibration-curve-free isotope dilution mass spectrometry(D-IDMS) accounting for species' transformations and conjugates, andachieving mass balance higher accuracy for actionable applications. Theinvention creates an accurate assessment of GSH, GSSG, conjugated GSH(CGSH), and GSH/GSSG ratio with corrections of transformations by directIDMS (D-IDMS) or direct speciated isotope dilution mass spectrometry(D-SIDMS) accounting for the four forms of glutathione that arefrequently referred to collectively and also quantified as GSH by mostother methods, without the essential and diagnostically significantcorrections taught herein.

Therefore, the present invention is a diagnostic and prognostic assayfor glutathione species concentrations in the blood or other fluids andtissues or matter of an animal or human for which diagnosis or prognosisis indicated, comprising assaying a quantity of blood and/or bloodcomponents or other fluids such as urine and tissues or matter such asimproved “GSH Assay” of drug conjugates with glutathione using specialdirect algorithmic speciated isotope dilution mass spectrometry(D-SIDMS) and algorithmic corrective evaluations. In some instances inAutism Spectrum of Diseases (ASD), the level of total Glutathione (tGSH)as defined herein is elevated. When blood is the sample for which assayanalysis is conducted, tGSH is measured at approximately 2.8-3.0 andgreater micromoles tGSH per gram blood, yielding a biochemical diagnosesthat indicate a 15% onset probability for ASD. For reduced glutathione,or GSH as defined herein, 2.2 to 2.4 micromoles per gram concentrationin blood, then a biochemical diagnosis of 40% probability of onset ofASD is indicated.

As a non-limiting example, a newborn baby blood test for ASD by accurateassessment of total glutathione and its related suite of tGSH, GSH,GSSG, CGSH and GSH/GSSG ratio in whole blood, red blood cells, serum,plasma, lymphatic fluid, cerebrospinal fluid, saliva, tears, urine,cells or tissues can identify those newborns that are at greatest riskfor ASD and thus identify those candidates for early intervention bynutritional therapy and other therapeutic strategies.

Low glutathione levels as defined and disclosed herein are alsodiagnostic for diseases where immune system compromised ordysfunctional, such as without limitation the condition known as chronicfatigue syndrome, several forms of cancer and more than a dozen diseaseconditions where immune system is compromised.

The definition of the glutathione compounds and conjugates disclosedherein is extremely important. Therefore, the present invention a methodof measuring and calculating (by a computer with output to a user) tGSH(total glutathione very particularly defined) with sample preparationand assay methods that have been confirmed to provide reliable tGSH andrelated diagnostic assays in blood or tissue from a patient. Moreover,the method of accurate measurement and calculation of tGSH, GSH (reducedglutathione) and the ratio of GSH to oxidized glutathione (GSSG) hasenabled for the first time the identification of the diagnostic range ofthe ratio of GSH/GSSG as 15-200, wherein a human having a ratio withinthe range 15-200 is vulnerable to disease including but not limited toautism and chronic fatigue syndrome. An increase in this ratio overtreatment time for a patient indicates positive response to therapy—upto maximum ratio values of about 600-800 for healthy individuals. Inparticular, after accurate measurement and calculation according to thisspecification, a tGSH concentration of 2.8-3.0 micromoles tGSH per gramof blood is prognostic of autism in children and young adults, and a GSHlevel of 2.2-2.4 micromoles per gram of blood is prognostic of autism inchildren and young adults. The measurements are conducted usingspeciated isotope dilution mass spectrometry to assess particularvariants (reduced, oxidized, conjugated, etc.) of glutathione and thecalculations are conducted automatically, following mass spectrometry,to provide readouts of tGSH, GSH and the ratio of GSH/GSSG using thefollowing equations, where tGSH is the sum of GSH_(corrected),GSSG_(corrected) and CGSH, and the ratio is given by:

$\frac{GSH}{GSSG} = \frac{z}{\gamma}$

where:z=GSH_(corrected)=GSH_(SIDMS)+α+βα=[GSH

GSSG]β=CGSH=Σ_(i)CGSH₁+CGSH₂+ . . . CGSH_(i)γ=GSSG_(corrected) (GSSG−[GSH

GSSG])The mass spectrometry measurements and calculations are performed usinga computer and the results are presented as an output to a user, usuallya printout on paper or a readout on a computer screen, showing valuesfor tGSH, GSH and the ratio of GSH/GSSG.

The present invention may be understood, therefore, to be a diagnosticand prognostic assay for glutathione concentration in blood or othertissue or matter of animals or human beings for which diagnosis orprognosis is indicated, comprising assaying a quantity of blood and/orother tissue or matter for glutathione constituent concentrations usingD-SIDMS. High or low glutathione levels are also diagnostic for diseasesof the compromised immune system or immune system dysfunction, such aswithout limitation the condition known as “chronic fatigue syndrome” andseveral types of cancer. There are at least twenty diseases in whichaccurate tGSH, GSSG, CGSH and corrected GSH/GSSG ratios are diagnosticbiomarkers, but accurate measurements of these “species” has beenimpossible heretofore. These diseases include, without limitation,Autism, Breast cancer, Diseases requiring hemodialysis, Conditionsrequiring Enalapril treatment, AIDS, Ataxia telangiectasia, Lung cancer,Macular degeneration, Diabetes, Preeclampsia, Bile duct obstruction,Colon cancer (initial), Colon cancer (advanced), Coronary heart surgery,Retinopathy of prematurity, Preeclampsia, Diabetic preeclampsia, andAtherosclerosis.

As a nonlimiting example, a child's blood test for ASD by accurateassessment of tGSB as described herein can identify those children thatare at greatest risk for ASD and thus identify those candidates forearly intervention by nutritional, interventions, and physiciansupervised therapy.

While the diagnostic levels reported above are for blood levels of tGSH,the invention is also applicable as assays of animal or human tissue,fluids other than blood, or matter including but not limited tokeratinaceous fingernail samples or hair. Diagnostic levels in tissuesor matter other than blood must be extrapolated because they will varyproportionately from blood.

In addition to using D-IDMS to assay for tGSH, the invention embracesthe speciation assay, with D-SIDMS, of any or all of tGSH, GSH, GSSG, orthe GSH/GSSG ratio, along with optional concurrent assessment ofconcentration of conjugated toxins and toxicants, including but notlimited to methylmercury, inorganic mercury, cadmium and lead.

Accurate and clinically relevant analyses of glutathione levels inpatient samples, including blood, fluids, tissue or biological matterhave been possible for the first time by implementing D-SIDMS. Previousassays for glutathione or GSSG have been highly inaccurate, unreliableand unsuitable for use as diagnostic or prognostic information. By“D-SIDMS” it is meant the technology described in the following U.S.patents and patent application documents, each of which is incorporatedherein by reference: U.S. Pat. No. 6,790,673 entitled, “Speciatedisotope dilution mass spectrometry of reactive species and relatedmethods;” U.S. Pat. No. 5,414,259 entitled, “Method of speciated isotopedilution mass spectrometry;” and U.S. patent application Ser. No.11/952,471 entitled, “Solid phase and catalyzed enabled automatedisotope dilution and speciated isotope dilution mass spectrometry.”

D-IDMS and D-SIDMS techniques have been successfully applied for theanalyses of tGSH, GSH and GSSG (glutathione disulfide) and obtainaccurate concentration values for all three species, primarily on thebases of isotopic values of the natural abundant and enriched standardswithout using an external calibration curve. The techniques were used tomeasure one (by D-IDMS) or three (by D-SIDMS) of the species in theblood samples of autistic children and controls.

As shown in FIG. 1 and prior to the present invention, quantitationmethods listed in the scientific literature involved creation of acalibration curve and serial dilution of a standard at differentconcentrations within the expected range of the analyte in the samplemixture. Quantitative analysis of GSH and its oxidized form, GSSG,although highly desirable as a key diagnostic biomarker, has not beenreliable achieved by using external calibration, as evidenced by widedifference in reported tGSH/GSSG concentration values as shown in thePRIOR ART FIG. 1. Prior to tGSH analysis, traditional sample preparationtypically involved treating disulfide bonds with a reducing agent, suchas dithiothreitol (DTT). For analysis of GSH and GSSG, analyticalmethods have included LC separation with either fluorescence or massspectrometry detection. A derivatization agent, such as NEM, is oftenused during sample preparation to block the thiol group on GSH, therebyminimizing its conversion to GSSG.

The D-SIDMS technique described in this specification includes spikingthe sample with known amounts of the enriched analytes of interest (andpairs of related analytes), analyzing the sample by mass spectrometry,and determining concentrations and extent of species interconversion byanalyzing analyte peak ratios. In other words, the optimized D-SIDMStechnique involves spiking the sample with known amounts of the enrichedanalytes of interest (and pairs of related analytes), analyzing thesample by mass spectrometry, and determining concentrations and extentof species interconversion by analyzing analyte MS peak ratios.

An attempt to preserve a sample was discussed in a paper by others, inwhich the authors added NEM in the blood draw tube such that theformation of the GSH-NEM conjugate occurred in the tube immediately whenthe blood was drawn. The NEM significantly reduces GSH oxidation butdoes not stop it. By contrast, in this invention, isotopically enrichedGSH and GSSG, along with the NEM, are added to the blood draw tube orother fluid or sample collection containers, thereby enabling use ofD-SIDMS (US EPA Method 6800A). This approach allowed measurement andcorrection of GSH oxidation/conjugation, which typically occurs duringshipment of a sample to the laboratory from a remote location, therebyenabling one to obtain samples for analysis from around the globe. Thisimproved sample stabilization and spiking method for GHS/GSSG analysissuch that, even if some GSH does convert to GSSG in the presence of NEM,the correct concentrations of both analytes can still be obtainedbecause of the ability to correct for conversion of GSH to GSSG at thetime of analysis in the laboratory.

Analysis of samples described in this invention were made using the massspectrometer in positive mode, and the collision energy used for tandemMS purposes for the GSH-NEM complex (which resulted in the primaryproduct ion being one with the loss of pyroglutamate) was 12V, and thatfor GSSG (resulting in the loss of two pyroglutamates) was 25V. Thetransitions monitored for the first 6.5 minutes were 433.1 m/z→304.1 m/z(for the most abundant isotope from the natural abundant GSH-NEMmolecular ion), 434.1 m/z→305.1 m/z, 435.1 m/z→306.1 m/z, 436.1m/z→307.1 m/z (for the most abundant isotope from the isotopicallyenriched GSH-NEM molecular ion), 437.1 m/z→308.1 m/z, 438.1 m/z→309.1m/z, 439.1 m/z→310.1 m/z, 440.1 m/z→311.1 m/z, 441.1 m/z→312.1 m/z,442.1 m/z→313.1 m/z, 443.1 m/z→314.1 m/z, 613.1 m/z→355.1 m/z (for themost abundant isotope from the natural abundant GSSG molecular ion),614.1 m/z→356.1 m/z, 615.1 m/z→357.1 m/z, 616.1 m/z→358.1 m/z (forpossible GSSG molecular ion resulting from combination of one naturalabundant GSH and one enriched GSH), 617.1 m/z→359.1 m/z (for mostabundant isotope from isotopically enriched GSSG molecular ion), 618.1m/z→360.1 m/z, 619.1 m/z→361.1 m/z (for possible most abundant isotopefrom enriched GSSG from combination of two enriched GSH's), 620.1m/z→362.1 m/z, 621.1 m/z→363.1 m/z, 622.1 m/z→364.1 m/z, 623.1 m/z→365.1m/z, 624.1 m/z→366.1 m/z. The transitions monitored for the last 18.5minutes were all of the ones for GSSG (613.1 m/z→355.1 m/z (for the mostabundant isotope from the natural abundant GSSG molecular ion), 614.1m/z→356.1 m/z, 615.1 m/z→357.1 m/z, 616.1 m/z→358.1 m/z (for possibleGSSG molecular ion resulting from combination of one natural abundantGSH and one enriched GSH), 617.1 m/z→359.1 m/z (for most abundantisotope from isotopically enriched GSSG molecular ion), 618.1 m/z→360.1m/z, 619.1 m/z→361.1 m/z (for possible most abundant isotope fromenriched GSSG from combination of two enriched GSH's), 620.1 m/z→362.1m/z, 621.1 m/z→363.1 m/z, 622.1 m/z→364.1 m/z, 623.1 m/z→365.1 m/z,624.1 m/z→366.1 m/z). The resolving power used for both the first andthird quadrupoles (MS1 and MS3) was what Agilent termed “unit” (peakwidth 0.7 amu at half height). The retention time for the GSH-NEMcomplex was ˜3 minutes, and that for the GSSG was ˜8 minutes. Thecapillary voltage was 3500V, and the fragmentor voltage was 135V. Thedwell time was 50 ms for the first 6.5 minutes and 5 ms for the final18.5 minutes. The autosampler tray was kept at 4° C.

The general equation for D-IDMS is shown below.

$\begin{matrix}{C_{x} = {\left( \frac{C_{s}W_{s}}{W_{x}} \right)\left( \frac{A_{s} - {R_{m}B_{s}}}{{R_{m}B_{x}} - A_{x}} \right)}} & {{Formula}\mspace{14mu} 1}\end{matrix}$

In Formula 1, as shown above. A_(x)=abundance of isotope A in thenatural abundant analyte in the sample; B_(x)=abundance of isotope B inthe natural abundant analyte in the sample; A_(s)=abundance of isotope Ain the enriched analyte in the spike; B_(s)=abundance of isotope B inthe enriched analyte in the spike; C_(x)=concentration of the naturalabundant analyte in the sample, which is what the equation is solvingfor C_(s)=concentration of enriched analyte in the spike in μmol/g;W_(x)=weight of the sample; W_(s)=weight of the spike; andR_(m)=measured ratio of isotope A to isotope B (obtained from the massspectrometry measurement).

A sample calculation according to the previous Formula 1 is shown belowas Table I:

TABLE I A_(x) = abundance of 182.1 m/z product ion in the naturalabundant GSH in the sample (0.00445) B_(x) = abundance of 179.1 m/zproduct ion in the natural abundant GSH in the sample (0.879) A_(s) =abundance of 182.1 m/z product ion in the enriched GSH in the spike(0.880) B_(s) = abundance of 179.1 m/z product ion in the enriched GSHin the spike (0.00337) C_(x) = concentration of GSH in the sample(unknown) C_(s) = concentration of enriched GSH in the spike μmol/g(2.31) W_(x) = weight of the sample (0.0198 g) W_(s) = weight of thespike (0.0222 g) R_(m) = measured ratio of 182.1 m/z product ion to179.1 product ion (obtained from the mass spectrometry measurements)(1.07) C_(x) = [((2.31 μmol/g)(0.0222 g))/(0.0198 g)] × [((0.880) −(1.07 × 0.00337))/((1.07 × 0.879) − 0.00445)] = 2.42 μmol/g Molecularweight of GSH = 307 μg/μmol C_(x) = in μg/g (or ppm) = (2.42 μmol/g)(307μg/μmol) = 742 ppm

In describing the D-SIDMS equations for GSH and GSSG analysis that aredesigned to correct for conversion of GSH to GSSG, it is best to startoff with a simple analogy. If one has several slices of wheat bread andseveral slices of white bread, one can make three types of sandwiches:ones with two slices of wheat bread, ones each with a slice of wheatbread and a slice of white bread (“hybrid” sandwiches), and ones withtwo slices of white bread. In a similar manner, if natural abundant GSH(307 g/mol) and isotopically enriched GSH with two ¹³C's and one ¹⁵N(310 g/mol) are equilibrated and oxidation occurs, one can end up withthree types of oxidized GSSG: natural abundant GSSG (612 g/mol) from twomolecules of natural abundant GSH (analogous to wheat bread sandwiches),partially enriched GSSG (615 g/mol) with two ¹³C's and one ¹⁵N from thecombination of one natural abundant GSH molecule and one isotopicallyenriched GSH molecule (analogous to the “hybrid” sandwiches), and GSSGwith four ¹³C's and two ¹⁵N's (618 g/mol) from two molecules of enrichedGSH (analogous to the white bread sandwiches). One can then determinehow much of the GSH converted to GSSG by spiking the sample withenriched GSSG in which the enrichment is different from the enrichedGSSG that comes from oxidation (such as GSSG that has four ¹³C's (616g/mol).

Suppose one has 1 mole of wheat bread slices (6.02×10²³ slices),referred to as “n” and the same number of white bread slices, referredto as “m,” that gets converted to sandwiches. Mathematical relationshipspertaining to the number of each kind of formed-sandwiches are describedbelow.

φ=# of ways to select k items from w=w!/[k! (w−k)!]e.g. if w=9 and k=2,φ=(9×8×7×6×5×4×3×2×1)/(2!)(7×6×5×4×3×2×1)=(9×8)/2=26If w is an extremely large number (such as 6.02×10²³) and k=2, for allpractical purposes, Φ=w²/2

The  total  number  of  sandwich  combinations = [(n + m)!]/k!((n + m) − k)!] = [(6.02 × 10²³ + 6.02 × 10²³)(6.02 × 10²³ + 6.02 × 10²³ − 1)]/2 = [(12.04 × 10²³)(12.04 × 10²³ − 1)]/2(neglect-1) = (12.04 × 10²³)²/2 = 7.25 × 10⁴⁷  sandwiches = (n + m)²/2The  probability  (expressed  as  a  decimal)  of  getting  a  wheat  bread  sandwich = [(n²)/2]/[(n + m)²/2] = [(6.02 × 10²³)²/2]/[(12.04 × 10²³)²/2] = (6.02 × 10²³)²/(12.04 × 10²³)² = 0.250The  probability  (expressed  as  a  decimal)  of  getting  a  white  bread  sandwich = [(m²)/2]/[(n + m)²/2] = [(6.02 × 10²³)²/2]/[(12.04 × 10²³)²/2] = (6.02 × 10²³)²/(12.04 × 10²³)² = 0.250The  probability  (expressed  as  a  decimal)  of  getting  a  sandwich  with  one  slice  of  wheat  bread  and  one  slice  of  white  bread = (n × m)/[(n + m)²/2] = 2  mn/(n + m)² = [(2)(6.02 × 10²³)(6.02 × 10²³)]/(6.02 × 10²³ + 6.02 × 10²³)² = 0.500The  same  0.500  answer  can  be  obtained  by  using  units  of  moles  instead = [(2)(1  mole)(1  mole)]/(1  mole + 1  mole)² = 0.500

These mathematical relationships can be applied to the analysis ofmolecules that are prone to dimerization and have most (over 80%) of theisotopic abundance coming from only one isotope, such as naturalabundant GSH (over 80% of isotope abundance is from the 307.1 g/molisotope) and isotopically enriched GSH (over 80% of abundance is fromthe 310.1 isotope). Let “n”=the μmol of natural abundant 307 g/mol GSHthat became oxidizied either to natural abundant 612 g/mol GSSG or“hybrid” 615 g/mol GSSG. Let “m”=the μmol of isotopically enriched 310g/mol GSH that became oxidized to either “hybrid” 615 g/mol GSSG orfully enriched 618 g/mol GSSG. The ratio of the probability of getting“hybrid” 615 g/mol GSSG (sandwich with one slice of wheat bread and oneslice of white bread) to the probability of getting fully enriched 618g/mol GSSG (sandwich with two slices of white bread) equals the ratio ofthe peak areas from LC/MS/MS analysis of the peaks in the chromatogramrepresenting those species of GSSG (after taking into account theabundances of those isotopes and subtracting out the contribution comingfrom natural abundant GSSG).

In positive mode ESI-MS, the most abundant isotope from natural abundant612 g/mol GSSG is detected at 613 m/z, that from “hybrid” 615 g/mol GSSGwith two ¹³C's and one ¹⁵N is detected at 616 m/z, that from enriched616 g/mol GSSG with four ¹³C's is detected at 617 m/z, and that fromenriched 618 g/mol GSSG with four ¹³C's and two ¹⁵N's (which came fromtwo 310 g/mol GSH's that each had two ¹³C's and one ¹⁵N) is detected at619 m/z. In positive mode electrospray tandem mass spectrometry(ESI-MS/MS) with the collision energy adjusted so as to make the mostabundant product ion that which results from the loss of twopyroglutamates from GSSG (loss of 258 g/mol), the numbers above changeto 355 m/z, 358 m/z, 359 m/z, and 361 m/z, respectively. Themathematical relationship described immediately following can be used todetermine GSH and GSSG concentrations.

${\left( {358\mspace{14mu} m\text{/}z\mspace{14mu} {peak}\mspace{14mu} {area}} \right)/\left( {361\mspace{14mu} m\text{/}z\mspace{14mu} {peak}\mspace{14mu} {area}} \right)} = {{\left( {{probability}\mspace{14mu} {of}\mspace{14mu} {getting}\mspace{14mu} {``{hybrid}"}\mspace{14mu} 615\mspace{14mu} g\text{/}{mol}\mspace{14mu} {GSSG}} \right)/\left( {{that}\mspace{14mu} {of}\mspace{14mu} {getting}\mspace{14mu} 618\mspace{14mu} g\text{/}{mol}\mspace{14mu} {GSSG}} \right)} = {{\left\lbrack {\left( {2\mspace{14mu} {mn}} \right)/\left( {n + m} \right)^{2}} \right\rbrack/\left\lbrack {\left( m^{2} \right)/\left( {m + n} \right)^{2}} \right\rbrack} = {2\mspace{14mu} n\text{/}m}}}$m= µmol  of  310  g/mol  GSH  that  became  oxidized = (2 × µmol  of  618  g/mol  GSSG  present) + (µmol  of  615  g/mol  GSSG  present)

The mass of the solution of the enriched 310 g/mol GSH that was added isknown, as is the mass and concentration of the enriched 616 g/mol GSSGsolution. Once can then determine the concentration (and, thus, μmol) ofthe 618 g/mol GSSG and 615 g/mol GSSG present by performing D-IDMS onboth with the 616 g/mol GSSG representing the enriched analyte. One μmolof 618 g/mol GSSG comes from two μmols of 310 g/mol GSH, and one μmol of615 g/mol GSSG comes from one μmol each of 307 g/mol GSH and 310 g/molGSH. Using this relationship, one can determine “m” (the μmol of 310g/mol GSH that became oxidized). Once “m” is known, one can determine“n” (the μmol of natural abundant 307 g/mol GSH that converted to either612 g/mol GSSG or 615 g/mol GSSG) using the following relationship:

[(358 m/z peak area)/((361 m/z peak area)*(2))]*m=n=μmol of 307 g/molGSH that became oxidized

Since the mass of the sample (e.g. blood sample, plasma sample, etc.) isknown, one can determine the concentration of GSH that became oxidizedto GSSG in units such as μmol/g (and, subsequently, μg/g) or smallerquantities, and perform the necessary subtraction from the uncorrectedGSSG concentration and the necessary addition to the uncorrected GSHconcentration.

The D-SIDMS analyses of the glutathione and its speciated compounds wereconducted on red blood cells, serum and plasma of children with ASD andcontrol children. This is one example of the diagnostic abilities andstatistical validation of the Glutathione Assay when measured as a suiteof compounds with corrections of inter-species transformations andconjugated forms provided by D-SIDMS.

Prior art associated with the measurement of GSH and its sub-compoundsand conjugates is in agreement that this type of measurement is notpossible with the currently available tools, methods or technologies.This invention overcomes these limitations by novel application ofD-SIDMS and mathematical, algorithmic derivation of analyteconcentrations using isotopic properties.

Referring now to FIG. 2, the average red blood cell total glutathioneconcentrations in children with autism and controls. The p value is0.011 with n=¢pairs. The significance is that this demonstrates that thehigher accuracy measurement is statistically significant as a biomarker.

In FIG. 3 the total glutathione reduced GSH is also a statisticallysignificant biomarker for diagnosing and evaluation of children withautism and undergoing treatment and is sensitive for trends and changesand responses during treatments. The control children show astatistically significant difference from the children with autism.

In FIG. 4, the importance of the relationship between sub-species ofglutathione are demonstrated as products and relationships ofsub-components of glutathione are statistically significant and withoutsub-components there would be no opportunity to either measure thesespecies accurately or to derive the significance of form the sub-speciesrelationships.

In FIGS. 5 and 6, Glutathione Logistic Regression Results have beenprepared that demonstrate the capability to use glutathione sub-speciestor predicting this disease. These logistic regression analysis ofglutathione concentrations which were performed take into account theregional prevalence of autism between 1 and 3 percent. The significantresults are shown in the figures relating glutathione concentrations toautism diagnostic percentage and also demonstrate the significance as abiomarker and diagnostic tool.

In practice, both drugs and toxins form conjugates with glutathione andhave been measured as demonstrated in FIGS. 7a-7b . The conjugates ofglutathione with methyl mercury and ethylmercury and inorganic mercuryappear as clustered peaks in FIGS. 7a -7 b.

In FIG. 8, the GSH and GSSG ratio of identical twin girls having ages ofless than eight years old were diagnosed to have ASD. The twin on theleft had a statistically significant increase in the GSH/GSSG ratioafter correction for GSH to GSSG oxidation in the testing procedure. Thetwin on the right after the same treatment did not have show theimprovement and indeed was reported medically not to have improved.These data demonstrate that the GSH/GSSG or zeta to gamma ratio iseffective at diagnosing immune function and changes in the condition ofthe patient.

It is also possible to quantify glutathione species according to theinvention for the purpose of tracking and quantifying heavy metal toxinswith which glutathione forms complexes. In other words, diagnosticlevels of tGSH, GSH and the GSH/GSSG ratio can be assessed and thencorrelated to GSH-conjugates containing heavy metals and theirconjugates, wherein such heavy metals may be, without limitation,methylmercury, inorganic mercury, cadmium, selenium, and lead. Oneskilled in the art who learns from the present invention how to measure,calculate and quantify glutathione and glutathione species will be ableto practice the invention to track and to quantify heavy metals byextension.

Although the invention has been described herein with particularity, theinvention is only to be limited insofar as is set forth in theaccompanying claims.

1-11. (canceled)
 12. A method of assessing toxicological properties in adrug candidate by performing a laboratory assay, said assay comprisingthe steps of: a) adding to a sample collection container all three of 1)isotopically enriched glutathione (GSH), 2) isotopically enrichedglutathione disulfide (GSSG) and 3) a measured amount of a thiol-groupblocking agent, followed by b) adding to said sample collectioncontainer of step a) an aliquot of a drug to be assessed for toxins,which drug forms a conjugate, said conjugate's significantly reducingbut not stopping the oxidation or transformation of said GSH in saidconjugate; c) assaying said aliquot for the ratio of GSH and GSSGoriginally present in said aliquot at the time of collection andcorrecting the assayed ratio for oxidation or transformation of said GSHby direct IDMS (D-IDMS) accounting for species' transformations andconjugates and direct speciated isotope dilution mass spectrometry bycalculating transitions based on the behavior of said isotopicallyenriched GSH and said isotopically enriched GSSG; and d) calculatingconjugated GSH (CGSH) for constituents of said aliquot, using theformulaCGSH=Σ_(i)CGSH₁+CGSH₂+. . . CGSH_(i) by adding an enzyme to the samplecollection container containing said aliquot of step b) and producingone or more data of chemically reactive metabolites of CGSHconcentrations which are calculated by a computer and output to a useraccording to the above calculations carried out on measurementsperformed by the mass spectrometer, wherein resulting mass spectrometrydata distinguish by cluster location benign forms of conjugates versustoxic forms.
 13. The method according to claim 12, wherein in step d)conjugates of inorganic mercury are apparent in any mass spectrometrydata of greater than 400 counts' intensity and m/z of 811-817 whereasone or more conjugates of methyl mercury, if any, are apparent inresulting mass spectrometry data of greater than 400 counts' intensityand m/z of 518-526 and one or more conjugates of ethyl mercury, if any,are apparent in resulting mass spectrometry data of greater than 400counts' intensity and m/z of 532-641.